Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosa causes irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol

Abstract

Iron is an essential nutrient for bacteria but the reactivity of Fe2+ and the insolubility of Fe3+ present significant challenges to bacterial cells. Iron storage proteins contribute to ameliorating these challenges by oxidizing Fe2+ using O2 and H2O2 as electron acceptors, and by compartmentalizing Fe3+. Two types of iron-storage proteins coexist in bacteria, the ferritins (Ftn) and the heme-containing bacterioferritins (Bfr), but the reasons for their coexistence are largely unknown. P. aeruginosa cells harbor two iron storage proteins (FtnA and BfrB), but nothing is known about their relative contributions to iron homeostasis. Prior studies in vitro have shown that iron mobilization from BfrB requires specific interactions with a ferredoxin (Bfd), but the relevance of the BfrB:Bfd interaction to iron homeostasis in P. aeruginosa is unknown. In this work we explore the repercussions of (i) deleting the bfrB gene, and (ii) perturbing the BfrB:Bfd interaction in P. aeruginosa cells by either deleting the bfd gene or by replacing the wild type bfrB gene with a L68A/E81A double mutant allele in the P. aeruginosa chromosome. The effects of the mutations were evaluated by following the accumulation of iron in BfrB, analyzing levels of free and total intracellular iron, and by characterizing the ensuing iron homeostasis dysregulation phenotypes. The results reveal that P. aeruginosa accumulate iron mainly in BfrB, and that the nutrient does not accumulate in FtnA to detectable levels, even after deletion of the bfrB gene. Perturbing the BfrB:Bfd interaction causes irreversible flow of iron into BfrB, which leads to the accumulation of unusable intracellular iron while severely depleting the levels of free intracellular iron, which drives the cells to an acute iron starvation response despite harboring “normal” levels of total intracellular iron. These results are discussed in the context of a dynamic equilibrium between free cytosolic Fe2+ and Fe3+ compartmentalized in BfrB, which functions as a buffer to oppose rapid changes of free cytosolic iron. Finally, we also show that P. aeruginosa cells utilize iron stored in BfrB for growth in iron-limiting conditions, and that the utilization of BfrB-iron requires a functional BfrB:Bfd interaction.

Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA

“…The Fdx, PaBfd from Pseudomonas aeruginosa (Figs. S1 and S2) was chosen as a template for the investigation, since this protein has shown to interact with a Bfr [24], and was also suggested to be involved in iron reduction and release from the Bfr. The phylogenetic study revealed one clade of…”

“…Fdxs have also been suggested to be involved in Fe 2+ release Bfr by direct protein-protein interaction [47], and a Fdx from Pseudomonas aeruginosa has been shown to interact with, and reduce, the iron core of PaBfr in vitro [64,24]. The interaction of Fdx and Bfr is thus established, but the proteinprotein interactions that allow the reduction of the iron-core of Dps are still not known.…”

Tyr25, Tyr58 and Trp133 ofEscherichia colibacterioferritin transfer electrons between iron in the central cavity and the ferroxidase centre

“…6 Their ability to lay down an iron-rich mineral inside a protein shell was one of the first indicators that ferritins had a role in iron storage, 4 though it is now not certain that is the primary function of all bacterioferritins. While bacterioferritin (BFR) in Pseudomonas aeruginosa does act as the general housekeeping store for iron, 7 in Escherichia coli it does not. 8 Both P. aeruginosa and E. coli contain another ferritin in addition to BFR, prokaryotic ferritin (Ftn), which is also a homopolymer of 24 subunits, 9 and it is Ftn that functions as the general housekeeping store for iron in E. coli.…”

Exploiting KRAS-driven Ferroaddiction in Cancer Through Ferrous Iron-Activatable Drug Conjugates (FeADC)

“…In a very recent GWAS study of lysosome function (Weber et al, 2020), it was shown that unloading of iron from transferrin and delivery of bioavailable ferrous iron (Fe 2+ ) to the cell was the single indispensable function of lysosomal acidity for cell proliferation. This remarkable finding was presaged by studies of the bacterium P. aeruginosa, where genetic deletion of a bacterioferrireductase (limiting cytosolic Fe 2+ ) produced acute iron starvation despite more than adequate Fe 3+ bacterioferritin stores (Eshelman et al, 2017). Elevation of cytosolic Fe 2+ may enable the survival of TKI-tolerant 'persister' cancer cells (Hangauer et al, 2017), which become uniquely reliant on the activity of GPX4, a lipid hydroperoxidase that protects from ferroptosis (Dixon and Stockwell, 2014;Stockwell et al, 2017).…”